Ultrasmall superparamagnetic iron oxide nanoparticles and uses thereof

ABSTRACT

The present invention provides a biomimetic contrast agent comprising an amine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core a targeting ligand attached to the nanoparticle core via a linker and an inert outer layer of a hydrophilic polymer conjugated to the targeting ligand and imaging methods using the biomimetic contrast agents. Also, provided is a dual functional contrast agent comprising a metal-doped iron (II) oxide/iron(III) oxide nanoparticle core, an inert layer of gold coating the nanoparticle core and a biodegradable cationic polymer linked thereto. The dual functional contrast agent is complexed to a therapeutic gene and when transfected into mesenchymal stem cells comprises a dual contrast agent and gene delivery system. In addition, methods of using the dual functional system are provided. Furthermore, kits comprising the biomimetic contrast agents and the dual contrast agent and gene delivery system are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims benefit of provisionalapplication U.S. Ser. No. 61/002,201 filed on Nov. 7, 2007.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of magnetic resonanceimaging and contrast agents and disease diagnosis and treatment. Morespecifically, the invention relates to iron oxide nanoparticles that areeither doped or not doped with varying amounts of metal ions and,optionally, gold-coated, and are surface-modified with a biomimetic andbioresponsive entity that imparts specificity of the contrast agent,which may include delivery of a therapeutic gene, to the desired target.

2. Description of the Related Art

In recent years, magnetic nanoparticles (MNPs) have generatedsignificant interest within the scientific community due to their hugerange of potential applications such as media materials for storagesystems (1-2), biotechnology (3-4), magnetic separation (5-6) targeteddrug delivery (7-8) and vehicles for gene and drug delivery (9-12).Among the various MNPs, undoped and transition metal-doped, e.g.,Magnetite or Maghemite, iron oxide nanoparticles, Fe₃O₄, g-Fe₂O₃ andM_(x)Fe_(y)O_(z) have found many applications in the area of biomedicaldiagnostics and therapy.

In the field of imaging, the superparamagnetic nature of iron oxidenanoparticles enables their use as potential contrast agents formagnetic resonance imaging (MRI). These nanoparticles with very largemagnetic susceptibilities strongly influence the T₁ and T₂ relaxation ofwater molecules surrounding these MRI contrast agents. In addition, themagnetic properties of these iron oxide nanoparticles are also dependenton particle size. Iron oxide nanoparticles that are below 15-20 nmretain superparamagnetism and influence the T₂ relaxation to the largestextent (13-14). This influence on the T₂ relaxation highly modulates theMRI properties of the iron oxide nanoparticles. Hence, by adjusting thecore size of the nanoparticles the magnetic properties for MRI can beenhanced. Typically, MNPs are synthesized with a particle size of 10-500nm in diameter for biomedical applications.

In addition to size, the biocompatibility, solubility, andmonodispersity of these MNPs are also critical for their use in vivo(15). However, the surface-chemical properties of MNPs do not facilitatethe conjugation of biomolecules. Therefore, the surface of these MNPsmust undergo modification or functionalization to enable the chemistryneeded for coupling biomolecules to MNPs. In other words, in order tomake the magnetic nanoparticles efficient delivery vehicles,introduction of suitable functional groups onto the surface of theparticle is essential so as to facilitate the conjugation of moleculesthat will increase its solubility and increase its availability forvarious conjugation processes. Also, the conjugation process has to beefficient, yielding a stable product, which does not compromise theactivity of the biomolecules. Moreover, the signal of the nanoparticlesfor imaging or other analytical techniques should not be affected as aresult of the conjugation. In addition, these nanoparticles should alsohave maximum surface area so as to facilitate the binding of a maximumnumber of linkage sites on the surface.

In the United States, breast cancer is second to lung cancer as theleading cause of cancer death in women. This year alone, ˜40,000 womenwill die from the disease despite a decline in the death rates.Detection is the best method to prevent mortality but only 60% ofcancers are detected at the earliest, subclinical stages of the disease.Also, ovarian cancer has the highest mortality rate of all gynecologicalmalignancies. Poor prognosis of the disease is directly related to latedetection after peritoneal tumor dissemination and the formation ofascites. Failure to detect early primary tumors or metastases results invery poor clinical outcome.

MRI is a powerful imaging modality for detection of cancer and otherdiseases because it provides high spatial resolution and excellent softtissue contrast. However, MRI imaging systems need to be developed thatare sensitive enough to detect cancers in the early stage of developmentand to detect metastatic cancers.

Thus, there is a recognized need in the art for improved contrast agentseffective to detect early stage primary and metastatic cancers viamagnetic resonance imaging, including contrast agents suitable todeliver a therapeutic gene during imaging. More specifically, the priorart is deficient in biomimetic contrast agents and a dual functioningcontrast agent/gene/drug delivery system effective to specificallytarget primary and metastatic cancer cells with MRI. The presentinvention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a biomimetic contrast agent. Thecontrast agent comprises an amine-functionalized iron (II)oxide/iron(III) oxide nanoparticle core, a targeting ligand attached tothe nanoparticle core via a linker and an inert outer layer of ahydrophilic polymer conjugated to the targeting ligand. The presentinvention is directed to a related biometric contrast agent furthercomprising a metal doping agent in the nanoparticle core. The presentinvention is directed to another related biometric contrast agentfurther comprising a gold coating on the nanoparticle core.

The present invention also is directed to a dual contrast agent thatcomprises a metal-doped iron (II) oxide/iron(III) oxide nanoparticlecore, an inert layer of gold coating the nanoparticle core and abiodegradable cationic polymer linked thereto. The present invention isdirected to a related contrast agent further comprising a DNA encodingan anti-tumor cytokine complexed with the cationic polymer.

The present invention is directed further still to a kit comprising thebiomimetic contrast agent described herein. The present invention isdirected further still to a kit comprising the dual contrast agentdescribed herein or the dual contrast agent complexed to a DNA encodinga therapeutic gene described herein. The present invention is directedto a related kit further comprising the buffers and reagents effectiveto transfect mesenchymal stem cells with the contrast agent-DNA complex.

The present invention is directed further still to a dual contrast agentand gene delivery system. The system comprises mesenchymal stem cells(MSCs) transfected with the dual contrast agent of described hereincomplexed with a DNA encoding an anti-tumor cytokine.

The present invention is directed further still to an in vivo methodusing magnetic resonance imaging for detecting an early stage primary ormetastatic cancer in a subject. The method comprises administering tothe subject a sufficient amount of the biomimetic contrast agentdescribed herein to provide a detectable contrast image of the contrastagent within the primary or metastatic cancer therein, wherein alocation of the image correlates to a location of the cancer.

The present invention is directed further still to a method for reducingmetastasis of tumor cells in a subject. The method comprisesadministering to the subject an amount of the mesenchymal stem cells(MSCs) comprising the dual contrast agent and gene delivery systemdescribed herein sufficient to target metastatic tumor cells andsimultaneously delivering the contrast agent and the anti-tumor cytokinethereto. A magnetic resonance image of the contrast agent within themetastatic tumor cells is obtained; where simultaneously imaging thecontrast agent and delivering the anti-tumor cytokine locates a site ofmetastatic tumor cells and induces a pro-inflammatory response againstthe same, thereby reducing metastasis of the tumor cells. The presentinvention is directed to a related method further comprisingimplementing one or both of a surgical regimen or one or more otherchemotherapeutic regimens.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention are briefly summarized. The above may be betterunderstood by reference to certain embodiments thereof which areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted; however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1B depict the syntheses of Gadolinium-doped UltrasmallParamagnetic Iron Oxide Nanoparticles (GdUSPIO) (FIG. 1B) andamine-functionalized Gadolinium-doped Ultrasmall Paramagnetic Iron OxideNanoparticles (amine-GdUSPIO) (FIG. 1B).

FIGS. 2A-2C are the FTIR spectrum of USPIO and amine-USPIO (FIG. 2A) andthe TEM image of USPIO (FIG. 2B) and amine-USPIO (FIG. 2C).

FIG. 3 shows results for the ninhydrin assay for the quantitativedetermination of the conjugation of 3-aminopropyltrimethoxysilane(APTMS) to ultra small paramagnetic iron oxide nanoparticles

FIGS. 4A-4C depict the synthesis of gold coated doped iron oxidenanoparticles (FIG. 4A) and the electronic absorption spectra of variousgold coated doped iron oxide nanoparticles (FIG. 4B) and TEM images ofgold-coated doped nanoparticles (FIG. 4C).

FIG. 5A-5D depict the Fourier Transform-infrared (FT-IR) vibrationalspectra of synthesized amine-GdUSPIO (top) and GdUSPIO (bottom)nanoparticles (FIG. 5A), the X-Ray Diffraction (XRD) pattern ofsynthesized GdUSPIO (top) and amine-GdUSPIO (bottom) nanoparticles (FIG.5B) and the Transmission Electron Microscopy (TEM) images of synthesized(FIG. 5C) GdUSPIO and (FIG. 5D) amine-GdUSPIO nanoparticles.

FIGS. 6A-6E depict the ¹H NMR spectra of homobifunctionalCOOH-PEG400-COOH diacid (b), the HPLC spectra for separation ofbioresponsive peptide with the RGDS (GRGDSGPQGLAG) ligand on ananalytical column (b) and a preparative column showing the main peaks ofinterest at 25 and 19 min (FIG. 6C) along with smaller peaksrepresenting byproducts of the synthesis reaction, the ¹H NMR spectrumof synthesized COOH-PEG₂₀₀₀-OMe outer stealth layer showing proton peaksof the polymer (FIG. 6D), the relaxivities for ultra small paramagneticiron oxide nanoparticles (r=130.8 mMol/lsec), amine-functionalized ultrasmall paramagnetic iron oxide nanoparticles (r=5.5 mMol/lsec) andinternal control CuSO₄ (r=1.2 mMol/lsec) (FIG. 6E).

FIGS. 7A-7E depict synthetic schema for amine-functionalized iron oxidenanoparticles (FIG. 7A) HOOC-TEG-t-butyl acrylate synthesis (FIG. 7B),peptide synthesis (FIG. 7C), USPIO-TEG-COOH synthesis (FIG. 7D), andUSPIO-TEG-Peptide-mPEG₅₀₀₀ synthesis (FIG. 7E).

FIGS. 8A-8F depict the NMR spectra of OH-TEG-Ots (FIG. 8A), OH-TEG-N₃(FIG. 8B), t-butyl ester-TEG-N₃ (FIG. 8C), NH₂-TEG-t-butylester (FIG.8D), t-butylester-TEG-COOH (FIG. 8E), and the MALDI-TOF data for dualbioresponsive peptide (FIG. 8F).

FIG. 9 is a schematic representation of IL-12 protein fromnanoparticle-transfected MSCs homing to tumor cells.

FIGS. 10A-10B depict the TEM image of synthesized Au-coated 4%Gd[III]-doped iron oxide nanoparticles where scale=20 nm (FIG. 10A) andthe MRI images of new contrast agents; Samples 1-7=CUSO₄; 8-9=4%,10-11=0%, and 12-13=2% iron oxide-doped Gd nanoparticles (FIG. 10B).

FIG. 11 depicts the ¹H NMR spectrum of synthesized reducible LLC-N-bocconjugated polymers.

FIG. 12 depicts the gel retardation assay of deprotected reducible LLCcopolymers: 1=free DNA; 2=linear PEI; 3=branched PEI; 4=PLL; 5=1/1;6=5/1; 7=10/1; 8=15/1; 9=20/1; 10=25/1; 11=30/1; 12=40/1; 13=50/1; and14=100/1 N/P ratios.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

As used herein “another” or “other” may mean at least a second or moreof the same or different claim element or components thereof. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

As used herein, the term “subject” refers to any recipient of thecontrast agents provided herein.

In one embodiment of the present invention there is provided abiomimetic contrast agent, comprising an amine-functionalized iron (II)oxide/iron(III) oxide nanoparticle core; a targeting ligand attached tothe nanoparticle core via a linker; an inert outer layer of ahydrophilic polymer conjugated to the targeting ligand. Further to thisembodiment the biomimetic contrast agent comprises a metal doping agentin the nanoparticle core. Examples of a metal doping agent aregadolinium, manganese or cobalt. In another further embodiment thebiomimetic contrast agent comprises a gold coating on the nanoparticlecore.

In all embodiments the ratio of the iron (II) to the iron(III) in thenanoparticle core may be about 1:1. Also, in all embodiments the linkermay comprise a triethylene glycol polymer or a polyethylene glycolpolymer. In addition, the inert outer layer may comprise a polyethyleneglycol polymer.

Also, in all embodiments the targeting ligand may comprise a contiguouspeptide sequence of an enzyme cleavable sequence and a targetingsequence in tandem. Further to these embodiments the targeting ligandmay comprise a blocking agent bound to a terminal sidechain amino acidof the peptides. In an aspect of these embodiments the enzyme cleavablesequence may be cleavable by an enzyme associated with ovarian cancercells. For example, the enzyme cleavable sequence may be ametalloproteinase-13 cleavable sequence, particularly the sequence shownin SEQ ID NO: 1. In another aspect the targeting sequence may be anendothelin-1 (ET-1) sequence, particularly the sequence shown in SEQ IDNO: 2, an integrin-binding sequence shown in SEQ ID NO: 3, or a humanimmunodeficiency virus Tat peptide sequence shown in SEQ ID NO: 4. Alsoin this aspect, the targeting ligand may have the contiguous sequencesshown in one of SEQ ID NOS: 5-7.

In a related embodiment the present invention provides a kit comprisingthe biomimetic contrast agent as described supra.

In another embodiment the present invention provides an in vivo methodusing magnetic resonance imaging for detecting an early stage primary ormetastatic cancer in a subject, comprising administering to the subjecta sufficient amount of the biomimetic contrast agent described supra toprovide a detectable contrast image of the contrast agent within theprimary or metastatic cancer therein, wherein a location of the imagecorrelates to a location of the cancer. Representative cancers may beovarian cancer or breast cancer or other cancer expressing MMP-13enzyme.

In yet another embodiment the present invention provides a contrastagent, comprising a metal-doped iron (II) oxide/iron(III) oxidenanoparticle core; an inert layer of gold coating the nanoparticle core;and a biodegradable cationic polymer linked thereto. Further to thisembodiment the contrast agent comprises DNA encoding an anti-tumorcytokine complexed with the cationic polymer. In both embodiments theanti-tumor cytokine may be interleukin-12. Also, the doping metal may begadolinium (III), manganese, cobalt, or ruthenium. In addition, thecationic polymer may comprise disulfide-reducible, linear,L-lysine-modified copolymers (LLC).

In a related embodiment the present invention provides a kit comprisingthe dual contrast agent described supra or this dual contrast complexedto a DNA encoding an anti-tumor cytokine. Further to this relatedembodiment the kit comprises the buffers and reagents effective totransfect mesenchymal stem cells with the contrast agent-DNA complex.

In another related embodiment the present invention provides a dualcontrast agent and gene delivery system, comprising mesenchymal stemcells (MSCS) transfected with the contrast agent described supracomplexed with a DNA encoding an anti-tumor cytokine. An example of ananti-tumor cytokine is interleukin-12.

In yet another embodiment of the present invention there is providedmethod for reducing metastasis of tumor cells in a subject, comprisingadministering to the subject an amount of the mesenchymal stem cells(MSCs) comprising the dual contrast agent and gene delivery systemdescribed herein sufficient to target metastatic tumor cells;simultaneously delivering the contrast agent and the anti-tumor cytokinethereto; and obtaining a magnetic resonance image of the contrast agentwithin the metastatic tumor cells; wherein simultaneously imaging thecontrast agent and delivering the anti-tumor cytokine locates a site ofmetastatic tumor cells and induces a pro-inflammatory response againstthe same, thereby reducing metastasis of the tumor cells. Further tothis embodiment the method comprises implementing one or both of asurgical regimen or one or more other chemotherapeutic regimens.

The present invention provides biomimetic ultrasmall paramagnetic ironoxide (USPIO) nanoparticles (USPIO) with dual bioresponsive elementseffective to selectively target tumor cells in the preclinical stage ofdevelopment for early detection of cancer for use in magnetic resonanceimaging. Such contrast agents act as early detection probes with broadavailability to patients and lead to life saving early detection andtreatment of cancer. The USPIO nanoparticles enhance sensitivity duringmagnetic resonance imaging (MRI) because of increased specificity forthe target by providing a modifiable surface. The presence of two levelsof targeting, based on specific tumor biochemistry, significantlyincreases the targeting of the probe to the tumor that would overcomethe hurdles of late detection, safety, rapid elimination, andnon-specific extravasation.

The contrast agent comprises an amine-functionalized nanoparticle coreof an undoped or metal-doped iron oxide, a linker, a targeting ligandand an outer inert or stealth layer. The nanoparticle core may be anFe(II)/Fe(III) oxide, such as which is biodegradable. The nanoparticlesmay be about 10-100 nm, where ultrasmall nanoparticles are less than 50nm, preferably about 20 nm to about 50 nm in diameter. For example,Fe(II)/Fe(III) oxide may be present in the core in a 1:1 ratio. Asuitable doping metal is, but not limited to, gadolinium, manganese,cobalt, or ruthenium. The linker may comprise a short polyethyleneglycol polymer (PEG), for example, a PEG₄₀₀ polymer, or a triethyleneglycol polymer. Optionally, the nanoparticle core may be coated with aninert layer of gold.

The targeting ligand is covalently attached, conjugated or linked to thenanoparticle core via the linker. The targeting ligand comprises twocontiguous peptide targeting sequences. The first targeting peptidesequence is cleavable by an enzyme associated with a cancer. Forexample, the cancer may be any cancer expressing themetalloproteinase-13 enzyme, such as ovarian cancer, and the peptide maybe cleavable by metalloproteinase-13 enzyme. This peptide sequence maycomprise a PQGLA sequence (SEQ ID NO: 1). The second targeting peptidesequence is a cancer cell specific peptide that binds to the tumorcells. For example, the second targeting peptide sequence may be anendothelin-1 (ET-1) sequence, such as CSCSSLMDKECVYFCHLDIIW (SEQ ID NO:2) or an integrin-binding sequence, such as an RGDS peptide sequence(SEQ ID NO: 3) or a human immunodeficiency virus (HIV) Tat peptidesequence, such as YGRKKRRQRRR (SEQ ID NO: 4). The peptides comprisingthe targeting sequences may be blocked, that is, the amino acid sidechains comprising the peptides may be blocked by a blocking agentselective for the amino acid. The peptides are synthesized as describedin Example 3.

Thus, a contiguous targeting ligand sequence may comprise the MMP-13cleavable sequence PQGLA and the ET-1 sequence asGCSCSSLMDKECVYFCHLDIIWGPQGLAG (SEQ ID NO: 5), the integrin-bindingsequence as GRGDSGPQGLAG (SEQ ID NO: 6), and the HIV Tat sequence asGYGRKKRRQRRRGPQGLAG (SEQ ID NO: 7). Glycine residues are added asspacers at the beginning and end of the contiguous sequence and betweenthe first and second targeting sequences to increase enzyme recognitionof the sequence. The nanoparticle-targeting ligand conjugate furthercomprises an inert or stealth outer layer, such as a hydrophilicpolymer, for example, but not limited to, a polyethylene glycol polymerconjugated to the targeting ligand. The PEG polymers may be a PEG₂₀₀₀ toabout PEG₅₀₀₀.

As a representative example, the contrast agents disclosed herein takeadvantage of the presence and role of metalloproteinase-13 (MMP-13) inovarian cancer and other cancers, such as breast cancer, because thisenzyme is active within these carcinomas that also express specificcellular receptors. The endothelin receptor (ET_(A)R) and endothelin-1(ET-1) peptide are produced in both primary and metastatic ovariantissue as well as the integrin (α_(v)β₃) receptors that bind thearginine-glycine-aspartic acid-serine (RGDS) sequence. In addition, theHIV Tat protein transduction domain has been shown to be a veryeffective and promiscuous cell penetrating peptide.

In general, upon administering the biomimetic contrast agent to asubject, the outer layer prevents non-specific cellular uptake of thecontrast agent as well as to limit premature interactions with blood andimmune components. At the first level of targeting, the enzyme cleavablepeptide sequence is recognized and cleaved only within the tumorcompartment by the enzyme which releases the outer protective stealthlayer. Release of the stealth layer reveals, as the second level oftargeting, the second peptide sequence, which as a ligand, specificallybinds to the tumor cells for cellular uptake of the contrast agents formagnetic resonance imaging.

The present invention also provides a dual contrast agent and genedelivery system useful with a MRI imaging modality. The system comprisesa nanoparticle-based platform (USPIO) to enable detection and cell-basedgene therapy treatment of metastatic cancer, such as, but not limitedto, metastatic breast cancer. The contrast agent may comprise thegold-coated metal-doped iron oxide nanoparticle core described herein.Particularly, a gadolinium-doped, such as gadolinium (III), iron oxidenanoparticle core is coated with an inert layer of gold.

The gold-coated nanoparticles are conjugated to cationic polymers thatmay comprise novel disulfide-reducible, linear, L-lysine-modifiedcopolymers (LLC). The cationic polymers are effective to form a complexwith a therapeutic gene, particularly a chemotherapeutic gene. Examplesof therapeutic genes may be interleukins, such as, interleukin-12. It iscontemplated that plasmid DNA encoding the therapeutic gene is complexedwith the cationic polymer using well-known and standard molecularbiological techniques.

The cationic polymer-AuGdUSPIO nanoparticle conjugate further functionsin a gene or drug delivery system. These cationic nanoparticles cantransfect mesenchymal stem cells (MSCs) using standard molecularbiological techniques. MSCs demonstrate homing properties to tumorcells, have been used as gene delivery vehicles, have reducedimmunogenicity and are easy to expand and maintain in cell culture.

In addition, the present invention also provides kits comprising thecontrast agents, including the contrast agent complexed to a DNAencoding a therapeutic DNA. Also, a kit comprising the contrast agentuseful in the gene delivery system may further comprise those buffers,reagents, etc. standard in the art that are useful during transfectionof a mesenchymal stem cell with the DNA-contrast agent complex.

As such, the present invention provides in vivo magnetic resonanceimaging methods using the contrast agents described herein. Thesecontrast agents may be administered in amounts sufficient to produce adetectable magnetic resonance image in cancerous cells or tissues ortumors. These methods are well suited to detect early stage primary ormetastatic cancers, such as, but not limited to, ovarian or breastcancers, upon targeting thereto. One of ordinary skill in the art iswell-suited to determine amounts of the contrast agents to administer toa subject, the route of administration and the MRI imaging conditionsnecessary to obtain a useable detectable image. Furthermore, such earlydetection greatly enhances the prognosis of the subject.

Also, the present invention provides methods of simultaneously detectingone or more sites of metastatic cancer cells and reducing metastasis ofmetastatic cells distal to a primary cancer or tumor using the dualcontrast agent and gene delivery system described herein during MRI. Themethod is particularly useful in reducing metastasis of a breast cancerto or metastatic breast cancer cells in the bones, lung, liver, andbrain of the subject. It is contemplated that delivery of the anti-tumorgene, such as, interleukin-12 would induce a pro-inflammatory responseagainst the metastatic cancer cells. One of ordinary skill in the art iswell able to determine the dosage of the transfected MSCs based on thetype of metastatic cancer, the progression of the cancer, the health ofthe subject, etc. and the conditions for MRI are easily determined. Uponlocation of the metastatic cells in the subject, the methods furtherprovide for additional therapeutic intervention, such as, surgicalremoval of cancerous tissue and/or one or more additionalchemotherapeutic agent or drug regimens as are well-known and standard.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Synthesis of Metal- and Undoped Ultrasmall Paramagnetic IronOxide Nanoparticles USPION

The synthesis of undoped iron oxide nanoparticles and gadolinium,manganese, cobalt, and ruthenium doped iron oxide nanoparticles werecarried out using a modified procedure by Hong et al. (17) Formetal-doped particles, the concentration of the gadolinium ions in thefinal product was 0, 2, 4, 6, 8, and 10% and was 8% for both manganeseand cobalt to the total amount of the Fe³⁺/Fe²⁺ ions. Calculations weredone such that the concentration of Fe²⁺/Fe³⁺=1. Calculated amount ofFeCl₂.4H₂O (M.W: 198.81) and FeCl₃.6H₂O (M.W:270.3) and, for metal-dopednanoparticles, a stoichiometric amount of metal chloride salts wereweighed and dissolved in ultra-pure water. Table 1 shows the amounts ofFeCl₂.4H₂O, FeCl₃.6H₂O and gadolinium used in the nanoparticle core.

TABLE 1 Amount of Amount of FeCl₂•4H₂O FeCl₃•6H₂O 1.5 g Fe₃O₄ + 0 g Gd0.6348 g 0.8651 g (0%) (3.2 mmol) (3.2 mmol) 1.47 g Fe₃O₄ + 0.03 g Gd0.6221 g 0.8478 g (2%) (3.136 mmol) (3.136 mmol) 1.44 g Fe₃O₄ + 0.06 gGd 0.6094 g 0.8305 g (4%) (3.072 mmol) (3.072 mmol) 1.41 g Fe₃O₄ + 0.09g Gd 0.596 g 0.813 g (6%) (3.008 mmol) (3.008 mmol) 1.38 g Fe₃O₄ + 0.12g Gd 0.5841 g 0.7959 g (8%) (2.944 mmol) (2.944 mmol) 1.35 g Fe₃O₄ +0.15 g Gd 0.5713 g 0.778 g (10%) (2.88 mmol) (2.88 mmol)

After the complete dissolution of the reactants, the mixture wastransferred to a three neck round bottom flask in an inert atmosphere.The reaction mixture was then heated to 60° C. for about 15-20 minsfollowed by the addition of liquid ammonia to a rapidly stirringmixture. Additional ammonia was added to adjust the pH of the solutionin the range of 8-9, and the nanoparticles were allowed to grow foradditional 90 minutes. After 90 minutes the reaction mixture was splitin two 500 ml centrifuge tube and the nanoparticles obtained werecentrifuged for 20 mins at 5000 r.p.m. Supernatant liquid obtained wasdiscarded and nanoparticles obtained were washed 3 times with ultra purewater until it showed a negative test for the presence of chloride ions.After no more chloride ions were present, half of the particles weretransferred to a three neck round bottom flask with Ethanol/Water as thesuspending solvent for functionalization, while other half was stored inwater in the 500 ml centrifuge tube for further characterization.

Synthesis of Amine-Functionalized Gadolinium-Doped UltrasmallParamagnetic Iron Oxide (Amine-GdUSPIO) Nanoparticles

FIGS. 1A-1B are synthetic schema for Gd-doped USPIOs and amine-GdUSPIOs.The metal-doped and undoped USPIO nanoparticles obtained in the previousstep were transferred to a three neck round bottom flask and heated to60° C. for about 15 minutes. After 15 minutes, 15 mls of3-aminopropyltrimethoxysilane (APTMS) was added drop wise throughaddition funnel or with a syringe to the rapidly stirring mixture. Thereaction mixture was further heated at 60° C. for about an hour withconstant stirring to allow complete functionalization. After thecompletion of reaction, the reaction mixture was again transferred to500 ml centrifuge tube and the mixture was centrifuged for about 20minutes at 5000 r.p.m. The supernatant liquid was discarded andfunctionalized nanoparticles were washed three times with ethanolfollowed by washing with water, and finally the reaction mixture wasstored in water for further characterization. FIG. 2A shows the IRspectrum of USPIO and amine-USPIO. Whereas the particle size measure byTEM was observed to be around 8-10 nm (FIGS. 2B-2C). Results for thequantitative determination of the conjugation of APTMS to ultra smallparamagnetic iron oxide nanoparticle using a standard ninhydrin assayare shown in FIG. 3 and the values obtained with this assay are shown inTable 2.

TABLE 2 Samples Absrobance Blank Final Conc. Amine nmole/ml 0.173 0.0660.107 46.52173913 0.142 0.066 0.076 33.04347826 0.124 0.066 0.05825.2173913 0.091 0.066 0.025 10.86956522 Average 0.1325 0.066 0.066528.91304348 STDEV 0.034278273 0 0.03427827 14.90359696Synthesize Au-Coated M_(x)Fe₂O₃

FIG. 4A is the synthetic scheme for gold-coated doped USPIOs. 100 ml ofmetal (Gd, CO, or Mn) doped iron oxide nanoparticle was diluted to 1 mlwith water and the particles were suspended with 2 mls of 0.1 M citricacid. Once the particles were suspended it was followed by thesuccessive alternate addition of 1 ml of 1% HAuCl₄ and 2 ml ofNH₂OH.HCl. The addition of 1% HAuCl₄ was increased to 2 ml after 1^(st)two additions. The amount of 0.1M NH₂OH.HCl was maintained same for allthe five additions. After the solution turned faint purple or pink. Twomore iterations of HAuCl₄ were added to precipitate all the particleswhich were then separated using magnet.

UV visible absorption spectra in FIG. 4B shows ˜530-575 nm absorption ofenergy by the gold coated nanoparticles in various samples. The presenceof Gd, Mn and Co in various samples was characterized using ICP-AES.Table 3 shows the relative concentration of all the elements in varioussamples. Further, the concentration of Gd in most of the sample wasdisplayed as 0 due to very low solubility of these gold coated samplesinto the solution which resulted in overall decrease in the detectionlimit of gadolinium. These particles were characterized with theparticle size of 15-20 nm as shown in the TEM images in FIG. 4C.

TABLE 3 Samples Fe Gd Mn Co Au USPIO 118.20 0.00  4% Gd USPIO 114.400.38  6% Gd USPIO 109.54 0.41  8% Gd USPIO 101.50 9.20 10% Gd USPIO87.50 10.53 Gold Coated Samples  2% Gd USPIO 15.4 0.08 0 0 65.93  4% GdUSPIO 2.15 0 0 0 36.33  6% Gd USPIO 1.5 0 0 0 63.25  8% Gd USPIO 2.9 0 00 53.58 10% Gd USPIO 2.4 0 0 0 28.3  8% Mn USPIO 40.7 0 1.3 0 3.09  8%Co USPIO 36.06 0 0 1.12 3.97

EXAMPLE 2 Spectroscopic Characterization of GdUSPIO and Amine-GdUSPIONanoparticles Fourier Transform-Infra Red (IR) Vibrational Spectrum

To confirm the presence of the amine groups present on amine-GdUSPIO,FT-IR spectroscopy was used for both GdUSPIO and amine-GdUSPIO. TheFT-IR spectra were measured at room temperature with a Nicolet Avator360 FT-IR spectrophotometer (Varian, Palo Alto, Calif.). Sampleconcentrations in the pellets were ˜1 mg of sample/200 mg of KBr salt.The synthesized MNPs were pressed into KBr pellets to record the solidstate infrared spectra. To improve the signal-to-noise ratio, multiplescans (264) of each sample were collected and the slowly slopingbaselines were subtracted from the digitally collected spectra usingGRAMS/32 software package (Thermo Galactic, Inc. Salem, N.H.).

The combined FT-IR spectra of synthesized amine-GdUSPIO (FIG. 5A-top)and GdUSPIO (FIG. 5A-bottom) showed the spinel structure of GdUSPIO andamine-GdUSPIO with vibrational bands at 577 and 680 cm⁻¹, whichcorresponded to the stretching vibrations of magnetite (n(Fe—O)) havingdifferent symmetry. In addition, the vibrational band at 577 cm⁻¹ isattributed to the T_(2g) modes of vibrations and the vibrational band at686 cm⁻¹ is attributed to the presence of A_(1g) modes of vibrations ofthe Fe—O. These vibrations in the IR region confirmed the presence ofthe magnetite crystal lattice.

The surface functionalization of the magnetite lattice was confirmed bythe presence of the 2922 and 2957 cm⁻¹ vibrational bands in the IRspectrum of amine-GdUSPIO. These vibrational bands of amine-GdUSPIO canbe attributed to the symmetric (n_(s)(CH)), and asymmetric stretchingvibrations (n_(as)(CH)), respectively, which corresponds to the —CH₂present in the APTMS molecule. The absorption band at 2843 cm⁻¹ can beattributed to the symmetric stretching vibrations of (n_(s)(O—CH₃)) ofAPTMS. The amine functionalized GdUSPIO was confirmed by the presence ofthe amine symmetric ((n_(s)(NH)) and asymmetric (n_(as)(NH)) stretchingvibrations of —NH₂ at 3423 and 3393 cm⁻¹, respectively.

Finally, the presence of the symmetric stretching vibrations of thesilane groups (n_(s)(Si—O)) at 991 cm⁻¹ in amine-GdUSPIO confirmed thepresence of the silane molecules bonded to the metal oxide surface. Thisfurther confirmed the amine-functionalization of the GdUSPIOnanoparticles. The observed FT-IR vibrational frequencies for bothGdUSPIO and amine-GdUSPIO are also tabulated in Table 4 for bettercomparison.

TABLE 4 Assigments GdUSPIO (cm⁻¹) amine-GdUSPIO (cm⁻¹) n(Fe—O) 577 577n(Fe—O) 680 686 n_(s)(SiO) — 991 n_(s)(O—CH₃) — 2843 n_(as)(CH) — 2922n_(s)(CH) — 2957 n_(s)(NH) — 3423 n_(as)(NH) — 3393X-Ray Diffraction (XRD) Pattern

The phase morphology of the MNPs was identified from the XRD pattern ofthe nanoparticles that was obtained from the Siemen D5000 q/2qDiffractometer (Siemen, New York, N.Y.) using Cu Ka X-Ray line. Briefly,1.0 mg of sample was suspended in isopropanol to form a concentratedslurry, which was then finely ground with a mortar and pestle. Thesample was spread on to the sample holder to form a thin layer ofnanoparticles and the XRD pattern was recorded by varying 2q from15-77°. The tube voltage was kept at 40 kV and the current used was 30mA at all time points. All the spectral figures were prepared with theIGOR Pro (version 6.0) software. The reflection at 311 lattice plane wasused to estimate the average crystalline size by applying the Scherrermodel. The Scherrer model is expressed as Equation 1:d=0.9l/b cos q  Eq. (1)where l=0.154 nm for Cu K_(a) line; d is the crystalline diameter, b isthe full width at the half maximum of the 311 peak in radians, and q isthe peak position in radians.

The spinel structure of both GdUSPIO and amine-GdUSPIO magnetitenanoparticles was confirmed from their XRD pattern. The XRD pattern ofboth GdUSPIO and amine-GdUSPIO is shown in FIG. 5B. The XRD pattern forboth GdUSPIO and amine-GdUSPIO showed similar peaks for the pure phaseof magnetite. In addition, the particles do not display sharpdiffraction peaks, which are typically observed for amorphous andultrafine materials. An average crystalline size of 8.45 nm wascalculated using Eq. 1 for both GdUSPIO and amine-GdUSPIO nanoparticles.

Transmission Electron Microscope (TEM)

The particle size and the morphology of both GdUSPIO and amine-GdUSPIOwere characterized using TEM. A carbon coated TEM grid (Pelco® No. 160)was used for nanoparticle visualization via a transmission electronmicroscope (Jeol Transmission Electron Microscopy, JEM-2010, Tokyo,Japan). Briefly, 20 μl of the nanoparticles suspended in water waspipetted on to a carbon-coated grid. The sample was allowed to settle onthe grid for approximately 1 minute in a 100% humidified atmosphere. Thecarbon-coated grid was then washed with one drop of water, followed bythe addition of another 20 μl of the nanoparticles onto the carboncoated grid. The grids were then left for approximately 45 secondsbefore gently removing excess sample through drying with a filter paper.

The TEM images of the GdUSPIO and amine-USPIO showed the presence ofMNPs having a distinct spheroidal morphology and size. The particle sizeobserved for both GdUSPIO and amine-GdUSPIO was found to beapproximately 10-15 nm as shown in FIGS. 5C-5D, respectively. Thissuggests that modification of the nanoparticles does not affect theoverall size and morphology of the nanoparticles. The TEM images alsoshowed the agglomeration of the particles due to the particles beinghighly magnetic.

Inductively Coupled Plasma-Atomic Emission Spectroscopy: (ICP-AES)

The concentration of iron and gadolinium in all samples were measuredwith an inductively coupled plasma-atomic emission spectrophotometer(ICP-AES) instrument (Thermo, Waltham, Mass.). Briefly, 1.0 mg of theGdUSPIO and amine-GdUSPIO was digested in concentrated HCl for about 45mins to dissolve. Once the particles were completely dissolved, the HClwas evaporated on a hot plate at 110° C. for 30 mins. The solidnanoparticles were then redissolved by heating with 1.0 mL ofconcentrated HNO3. Once the sample was dissolved, the solution wasdiluted 10 times with water to a final concentration of approximately100 ppm. These solutions were finally used to obtain the ICP-AES data.

Elemental analysis by ICP-AES confirmed the presence of Gd ions in thenanoparticles. The ICP-AES data with respective concentrations in ppmand in moles for Fe, Gd, and Si are tabulated in Table 5. The elementalanalysis by ICP-AES also showed the relative Si in the samples. Theamount of the Gd in both 4 and 6% doped nanoparticles was found to beapproximately 0.4 ppm as compared to 114 and 109.54 ppm of iron,respectively. This suggested that the effective doping of the Gd in 4and 6% GdUSPIO is approximately 0.1%. Similarly, the amount of thedoping in 8 and 10% Gd-doped USPIO was found to be 3.2 and 4.2%,respectively. This also corresponds to the doping efficiency of 42% in10% GdUSPIO samples.

These discrepancies in the results can be attributed to the lowerdetection limit of the instrument for the amount of Gd present in thesample. The ICP-AES instrument can measure the Fe concentration in therange of 10-120 ppm, Hence, the samples were prepared considering themaximum concentration of 120 ppm for Fe²⁺/Fe³⁺ ions. However, thisdecreased the detection limit for the Gd ions as they were dopedstarting with the initial ratio of 4 and 6% and the actual doping waslower. Additionally, the molar ratio of the Gd in the sample decreased,as signal-to-noise ratio for the detection of Gd ions in the sampledecreased. This led to an inaccuracy in the detection of the Gd at thelower limit. Furthermore, the molar ratio of the amount of the Fe to theamount of the Gd for 10% GdUSPIO was calculated to be 1.56:0.066 ascompared to 2.98:0.02 shown before (16). The presence of the Si metal inamine-functionalized samples further confirmed theamine-functionalization of Gd-doped USPIO nanoparticles.

TABLE 5 Concentration in ppm Moles Sample Fe Si Gd Fe Si Gd USPIO 118.200.00 0.00 2.11 0 0 USPIO-amine 112.78 3.83 0.00 2.019 0.13 0  4% GdUSPIO 114.40 0.00 0.38 2.048 0 0.002  4% Gd USPIO-amine 147.20 3.14 0.572.63 0.11 0.0036  6% Gd USPIO 109.54 0.00 0.41 1.96 0 0.0026  6% GdUSPIO-amine 102.70 2.68 5.85 1.83 0.095 0.037  8% Gd USPIO 101.50 0.009.20 1.81 0 0.058  8% Gd USPIO-amine 113.14 3.06 9.38 2.02 0.10 0.05910% Gd USPIO 87.50 0.00 10.53 1.56 0 0.066 10% Gd USPIO-amine 90.72 2.927.72 1.62 0.10 0.049

EXAMPLE 3 USPIO-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀

Synthesis of Homobifunctional COOH-PEG₄₀₀-COOH Diacid

The homobifunctional PEG₄₀₀ diacid is synthesized according to thefollowing procedure. Briefly, 20 g of PEG₄₀₀ is dissolved in 300 mLacetone with vigorous stirring in an ice bath. After 15 minutes, 40 mLof Jones reagent (chromium oxide, sulfuric acid, and water) is slowlyadded and the reaction is stirred for 16 hours. After this time, 20 mLof isopropanol is added to quench the reaction and 2 g of activatedcarbon is added and stirred for 2 hours. After this time, the reactionmixture is filtered and the solvent is evaporated. The product isobtained from solvent extraction with methylene chloride (CH₂Cl₂) andcharacterized with ¹H NMR. FIG. 6A shows that PEG methylene (—CH₂)protons, and end methylene (—O—CH₂) protons of PEG₄₀₀ are observed atabout 1.35 ppm (a), 4.23 ppm (b) and 3.79 ppm (c) with no impuritiesobserved from the spectrum as expected.

Synthesis of Heterobifunctional HOOC-PEG₄₀₀-NHS

The homobifunctional PEG₄₀₀ diacid synthesized from the previous step (5g, 8.96 mmol) is reacted with DCC (3.69 g, 17.8 mmol) and NHS (0.896 g,8.96 mmol) in 100 mL of anhydrous methylene chloride (CH₂Cl₂) and thereaction is stirred for 24 hours. The reaction is quenched withultra-pure H₂O, filtered, and concentrated. The unreacted NHS is removedby solvent extraction with benzene and the polymers are precipitated inether. The product is then column chromatographed to obtain pureheterobifunctional COOH-PEG₄₀₀-NHS. The quantitative analysis of the NHSsubstitution is determined with ¹H NMR, MALDI-TOF and FT-IR.

Synthesis of Blocked Peptides (b-Peptides)

Each of the bioresponsive peptides—ET-1, the RGDS, and the HIV tatpeptide are synthesized in tandem with the MMP-13 cleavable sequence,PQGLA, to produce three separate contiguous peptides:GCSCSSLMDKECVYFCHLDIIWGPQGLAG, GRGDSGPQGLAG, and GYGRKKRRQRRRGPQGLAGrespectively, in which glycines (gly, G) is added as spacer amino acidsto increase enzyme recognition of the peptides. These peptides aresynthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry onan Applied Biosystems 431A peptide synthesizer (Foster, Calif.). Ascontrols, scrambled peptides are also synthesized and conjugated tonanoparticles in order to demonstrate specificity of the peptides forenzyme cleavage as well as receptor binding and cell penetration.

All peptides are synthesized with an ethylene diamine resin on theC-terminus of the peptides (synthesis occurs from the C— to N-terminus),which after cleaving the peptide from the resin introduces an aminegroup for conjugation to the carboxylate group of the PEG₄₀₀ linker.Moreover, all peptides are synthesized with ‘blocked’ amino acids inwhich all reactive side chains of the amino acids are protected.Selective blocking (different blocking groups) and deprotection of theN-terminal glycine facilitates conjugation of the peptide to the outerstealth PEG layer via the N-terminal amine group. After synthesis, theresin of each of the b-peptide is cleaved. Briefly, 800 mg of eachpeptide is dissolved in 25 mL of 3% trifluoacetic acid in CH₂Cl₂ and thereaction mixture is stirred for 4 hours. After this time, the reactionmixture is filtered to remove the cleaved resin. The cleaved peptidesare purified with reverse phase high pressure liquid chromatography asshown in FIGS. 6B-6C for GRGDSGPQGLAG (RGDS) containing peptide.

Synthesis of Ultra Small Paramagnetic Iron Oxide-PEG₄₀₀

Each of the synthesized bioresponsive peptidesGCSCSSLMDKECVYFCHLDIIWGPQGLAG, GRGDSGPQGLAG, and GYGRKKRRQRRRGPQGLAG areconjugated to ultra small paramagnetic iron oxide nanoparticlesseparately via a short (PEG₄₀₀) linker. Briefly, 1 g ofamine-functionalized ultra small paramagnetic iron oxide nanoparticlesare dispersed in basic water and stirred for 30 minutes under argon gas.After this time, a 3.0 mole excess of COOH-PEG₄₀₀-NHS is added slowlythrough an addition funnel and stirred overnight. The reaction mixtureis acidified, centrifuged to remove unreacted PEG₄₀₀, and washed withultra-pure water as previously described. The product is characterizedfor the absence of the amine groups and the presence of PEG₄₀₀ with aninhydrin assay and FT-IR spectroscopy. These pegylated nanoparticlesare then conjugated to the bioresponsive peptides.

Synthesis of Ultra Small Paramagnetic Iron Oxide-PEG₄₀₀-b-Peptide

Briefly, a 3.0 mole excess of RGDS, ET-1, and HIV Tat with MMP-13cleavable sequence or scrambled peptide sequence of each of thecontiguous peptides mentioned above are weighed separately and each aredissolved in 25 mL of CH₂Cl₂ and transferred to separate three-neckround bottom flasks purged with argon gas. After about 30 minutes, 0.5 gof the ultra small paramagnetic iron oxide-PEG₄₀₀ nanoparticles areadded to each of the reaction solutions, which are then stirredovernight. The reaction mixtures are then washed separately with waterand CH₂Cl₂ to remove unreacted peptide and each of the differentpeptide-conjugated nanoparticles are washed with 0.1M HCl to quench thereaction, followed with 0.1M NaOH for neutralization. The CH₂Cl₂ isdried with anhydrous MgSO₄, filtered, and the solvent is evaporated. Thedifferent peptide-conjugated nanoparticles are then characterized with astandard bicinchoninic assay (BCA) as per the manufacturer's instructionto determine the amount of peptides conjugated per nanoparticle.

Synthesis of Stealth Layer: Heterobifunctional COOH-PEG2000-OMe

Commercially available monomethoxy-PEG (HO-PEG₂₀₀₀-OMe) (MW 2000) isconverted to COOH-PEG₂₀₀₀-OMe and conjugated to the bioresponsivepeptides (from the N-terminus selectively deprotected glycine) to forman inert outer stealth layer that is released upon subsequent cleavageby MMP-13 enzymes in the tumor to reveal a targeting ligand.

The synthesis of COOH-PEG₂₀₀₀-OMe (outer stealth layer) is performedaccording to the following procedure. Commercially availablemonomethoxy-PEG (HO-PEG₂₀₀₀-OMe) (MW 2000) is converted toCOOH-PEG₂₀₀₀-OMe and conjugated to the bioresponsive peptides (viaN-terminus selectively deprotected glycine) to form an outer stealthlayer that is released upon cleavage by MMP-13 enzymes in the tumor toreveal a targeting ligand. This polymer is synthesized and characterizedexactly as described above. As can be seen from FIG. 6D, the carboxyl(—COOH), PEG methylene (—CH₂) protons, and end methylene (—O—CH₂)protons of PEG₂₀₀₀ are observed at 4.0 ppm, 3.6 ppm, and 3.3 ppmrespectively, with no impurities observed from the spectrum as expected.

Synthesis of Ultra Small Paramagnetic IronOxide-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀

Briefly, 0.5 g of each different ultra small paramagnetic ironoxide-PEG₄₀₀-b-Peptide nanoparticles are dissolved in 30 mL CH₂Cl₂ inseparate three-neck round bottom flasks purged with argon gas. After 30minutes, 25% piperidine in dimethylformamide is added to each reactionmixture that is stirred for 2 hours. After this time, a 3 mole excess ofCOOH-PEG₂₀₀₀-OMe is added to each of the reaction mixture, which isstirred for 4 to 6 hours. The solvent is then evaporated and theproducts are washed separately to remove unreacted mPEG₂₀₀₀. The finalbiomimetic ultra small paramagnetic iron oxide-PEG₄₀₀-Peptide-mPEG₅₀₀₀nanoparticles are obtained by deprotection of the conjugated peptides.Briefly, 0.5 g of each different ultra small paramagnetic ironoxide-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀ nanoparticles are dissolved in 30 ml 50%TFA in CH₂Cl₂ in separate three-neck round bottom flasks under argon gasfor 3 to 4 hours. After the reaction time, the nanoparticles arefiltered from the solvent containing the protecting groups, and thenanoparticles are washed several times with CH₂Cl₂. The finalnanoparticles are then dried in vacuo. These nanoparticles arecharacterized with TEM and dynamic light scattering to determine theparticle size and hydrodynamic volume respectively.

Quantitative Determination of the T2 Relaxivities

MRI experiment are performed on a 3.0 T horizontal bore General ElectricExcite HD imaging system, using the standard 8 channel head coil.Multi-Echo (n=8) Spin-Echo images are obtained for each sample set. TheTE values for the echo train are centered around the T2 valuesdetermined with a large TR=5000 s to exclude any influence of T1relaxation effects. The T2 relaxation times are calculated from a linearfit of the logarithmic region-of-interest signal amplitudes versus TEusing the Matlab (Mathworks Inc.) software. Relaxivity values areederived according to equation (1)

$\begin{matrix}{\frac{1}{T_{2}} = {\frac{1}{T_{20}} + {r \cdot c}}} & (1)\end{matrix}$with T₂₀ as the T2 value for the agarose gel, r as relaxivity and c asconcentration. Statistical analysis (ANOVA and Student's t test) areperformed on each data set.

Agarose phantom gels with and without the nanoparticles are prepared andanalyzed with an MRI scanner. Agarose gels are used in these studies tosimulate the protons present in a tissue environment. The controls forthe experiment include agarose gels only and commercially purchasedFerumoxsil® (Mallinckrodt, Inc., Hazelwood, Mo.) with a crystal size of10 nm.

A set of seven phantoms are built for these studies that contain agarosegels only, agarose gels with Ferumoxsil®, and agarose gels with varyingconcentrations of the following: synthesized ultra small paramagneticiron oxide nanoparticles, amine-functionalized ultra small paramagneticiron oxide nanoparticles, ultra small paramagnetic iron oxide-PEG₄₀₀nanoparticles, and each of the different ultra small paramagnetic ironoxide-PEG₄₀₀-Peptide nanoparticles, and ultra small paramagnetic ironoxide-PEG₄₀₀-Peptide-PEG₂₀₀₀ nanoparticles.

The T₂ values for each of the intermediate ultra small paramagnetic ironoxide nanoparticlesm e.g. ultra small paramagnetic iron oxide-PEG₄₀₀,are determined in order to fully characterize the relaxation of thenanoparticles at each modification step. Briefly, Ferumoxsil® and ironoxide nanoparticles at concentrations of 0.001, 0.01, 0.1, 1.0, 10.0,25.0, 50.0, 75.0, and 100 μg/mL are added to a boiled agarose solutionto form a final concentration of 1% agarose gel. The solution is mixedand quickly poured into sterile 15 mL polypropylene tubes that areslowly cooled to room temperature so as to avoid trapping air bubblesinside the tubes. The tubes are then placed in a polystyrene rack thatand are secured in a water bath fitted to a MRI head coil.

FIG. 6E shows relaxivities values of control ultra small paramagneticiron oxide nanoparticles, synthesized ultra small paramagnetic ironoxide nanoparticles (130.8 mmol/s), and amine-functionalized ultra smallparamagnetic iron oxide nanoparticles (5.5 mmol/s).

The enzymatic degradation of the nanoparticles with MMP-13(collagenase-3)-labile domains is assessed by monitoring the kinetics ofmass loss of the ultra small paramagnetic iron oxide nanoparticles as afunction of time and enzyme concentration. The cleavage of the MMP-13sequence within the nanoparticle results in the release of the outerstealth PEG layer that corresponds to a molecular weight of about 2000but produces nanoparticles with a smaller hydrodynamic volume. Thespecificity of the sequence for cleavage via MMP-13 is investigated withthe non-specific enzyme elastase.

Release Kinetics of Outer Peg Layer: MMP-13-Dependent Degradation ofBiomimetic USPIO Nanoparticles

The three different biomimetic ultra small paramagnetic iron oxidenanoparticles (ET-1, RGDS, HIV Tat) and their corresponding ultra smallparamagnetic iron oxide nanoparticles with scrambled peptide sequencesare weighed separately and incubated with varying concentrations ofMMP-13 or with elastase. Briefly, 100 mg of each iron oxidenanoparticles are added to separate vials each with 1.0 mL of phosphatebuffered saline (PBS) with 0.2 mg/mL of sodium azide and 1 mM calciumchloride (CaCl₂). Four sets of vials are labeled as 0, 8, and 24 hoursand 0.025, 0.0050, and 0.0025 mg/mL (collagenase) and 0.025 mg/mLelastase. Crude collagenase or elastase is then added to the vials toproduce the enzyme concentrations listed above. Control samples alsoinclude untreated biomimetic ultra small paramagnetic iron oxidenanoparticles. All samples are incubated at 37° C. and at predeterminedtime intervals stated above samples are removed and analyzed with HPLC,MALDI-TOF, and TEM to determine the molecular weight of the released PEGand the nanoparticle size for both treated and non-treated samples.Statistical analysis (ANOVA and Student's t test) is performed on eachdata set (N=3). Only nanoparticles containing the correct MMP-13cleavable sequence are cleaved by the enzymes to release the outerPEG₂₀₀₀ layer.

Receptor-Mediated Uptake of Biomimetic USPIO Nanoparticles with ET-1 andRGDS

Cells (OVCA and HUVECs) are seeded at a density of 9×10⁵ cells/well in a6-well plate and incubated at 37° C. for at least 24 hours prior totreatment with nanoparticles. The cells are untreated or treated withplain, bioresponsive, or scrambled nanoparticles at a concentration of0.03 μmol/mL. In addition, the cells are simultaneously treated withcrude collagenase to facilitate the release of the PEG stealth layer.Controls also include cells not treated with the enzyme. Afterincubation of the cells at predetermined time intervals of 0 hour, 4hours, 8 hours, and 24 hours, the cells are washed thrice with 1×PBS(0.1 mol/L, pH 7.4) buffer, trypsinized, centrifuged for 5 minutes at1500 rpm, and counted with a hemocytometer. A total of 7×10⁵ cells arethen mixed with 1% agarose at 37° C. and the T₂ relaxation values aremeasured with MRI. For the competition assay, either free ET-1 or RGDSligand at a 10,000:1 ratio (ligand:nanoparticles) is used to pretreatthe cells prior to the addition of the nanoparticles and T₂ values aredetermined.

Receptor-Independent Uptake of Biomimetic Ultra Small ParamagneticNanoparticles with HIV Tat

Cells (OVCA and HUVECs) are seeded, treated, and analyzed as describedabove except that the incubation times are performed at 4° C. and 37°C., to demonstrate that uptake of the biomimetic nanoparticles occurs ina receptor-independent manner in which receptor-mediated uptake isinhibited at 4° C.

For Prussian blue staining, cells are plated and treated with plain,bioresponsive, or scrambled nanoparticles as described above except thatglass coverslips is placed in the 6-well plates prior to seeding thecells and the cells are incubated for 24 hours. After incubation, thecoverslips are washed with PBS and fixed with the organic solventsmethanol and acetone. Briefly, the coverslips are added to cooled (−20°C.) in methanol for 10 minutes. The coverslips are then removed from themethanol and the cells are permeabilized with cooled acetone (−20° C.)for 1 minute. The coverslips are then added to 10% potassiumferrocyanide for 5 min and 10% potassium ferrocyanide in 20%hydrochloric acid for 30 minutes, and the nuclei are counterstained byadding the coverslip to a solution of propidium iodide at aconcentration of 0.01 mg/mL. The coverslips are then washed thrice with1×PBS buffer to remove excess propidium iodide and analyzed withmicroscopy to show uptake of the bioresponsive nanoparticles that areapparent as blue granules.

Cytotoxicity of Biomimetic Ultra Small Paramagnetic Iron OxideNanoparticles

Cells (OVCA and HUVECs) are seeded and treated as described above. Afterthe incubation times, the cells are treated with MTT with the method ofMossman. Briefly, the cells are washed with 1×PBS and then incubatedwith a solution of MTT in 1×PBS buffer at a concentration of 2 mg/mL.The plates are then incubated at 37° C. for 4 hours, after which themedium is removed and 1.0 mL of dimethylsulfoxide is added to the wellsto dissolve the formazan crystals. The absorbance of the samples isdetermined at 570 nm and the readings are normalized to that of theuntreated cells.

EXAMPLE 4 USPIO-TEG-Peptide-PEG Contrast Agent

The synthesis of biomimetic USPIO-TEG-Peptide-mPEG₅₀₀₀ is depicted inFIGS. 7A-7E.

Synthesis of HO-TEG-Ots

10 gms (0.0255 moles) of TEG was weighed in a 3 neck RBF purged withnitrogen. To the mixture 4.86 gms of Tosylchloride was added at 0° C.and the mixture was stirred for 3 hours. At the end of three hours 0.85mls of TEA was added to neutralize the acid formed and the reactionmixture was left to stir overnight. The progress of the reaction wasthen monitored by TLC. After the completion of the reaction the solventwas evaporated and the sample was dried under vacuum. The crude productwas then chromatographed using ethyl acetate as the solvent. Initialfractions were then left out and the fractions containing pure productwere then pooled together and solvent was evaporated and kept in vacuum.Sample obtained was 3.5 gms and NMR showed pure product as shown in FIG.8A.

Synthesis of HO-TEG-N₃

1.5 gms of HO-TEG-Ots was dissolved in 25-30 mls of DMF followed by theaddition of 0.007 gms of sodium azide. The reaction mixture was refluxedand progress of the reaction was monitored by TLC. The TLC showed theformation of the product and the completion of the reaction using 2%methanol in methylene chloride NMR in FIG. 8B shows the presence of thepure product.

Synthesis of N₃-TEG-t-butyl Ester

0.047 gms of potassium t-butoxide (M.W: 111.22, 0.425 mmoles) wasweighed under nitrogen and transferred to a 50 ml round bottom flaskcontaining 10 mls of anhydrous THF purged with N₂. After 30 mins, 1 gmof N₃O-TEG-OH (0.425 mmoles) was added slowly to the solution using asyringe. Reaction mixture was allowed to react under the nitrogenatmosphere for almost 3-4 hours. After all the solid went into thesolution, 0.064 gms t-butyl acrylate (M.W: 128.17, 0.5 mmoles) was addedslowly to the solution using a syringe. On the addition of t-butylacrylate the reaction temperature increased and care was taken tomaintain at ambient. The reaction mixture was allowed to stir overnight.The progress of the reaction was monitored by TLC. After the completionof the reaction, the solvent was evaporated and the oily residue alongwith the solid obtained was dissolved in mixture of ethyl acetate andwater (50:50) and layers were separated. The reaction mixture was thenpurified using column chromatography using Ethyl acetate/Hexane as thesolvent. Various fractions which seemed similar in TLC were pooledtogether and the product obtained was characterized by NMR. NMRindicated the presence of pure product (FIG. 8C).

Synthesis of NH₂-TEG-t-butyl Ester

1.5 gms of N₃-TEG-t-butyl ester was weighed under nitrogen andtransferred to a 50 ml round bottom flask containing 25 mls of anhydrousTHF purged with N₂. After 30 mins, 1.2 moles of triphenyl phosphine wasadded slowly to the reaction mixture. Reaction mixture was allowed toreact overnight under the nitrogen atmosphere. The progress of thereaction was monitored by TLC (Methanol/Ethyl Acetate 1/10). After thecompletion of the reaction, the solvent was evaporated and the oilyresidue was then purified using column chromatography. Various fractionswhich seemed similar in TLC were pooled together and the productobtained was characterized by NMR. NMR indicated the presence of pureproduct (FIG. 8D).

Synthesis of HOOC-TEG-t-butyl Ester

FIG. 7A is the synthetic scheme for amine-functionalized iron oxidenanoparticles. FIG. 7B is the synthetic scheme for HOOC-TEG-t-butylester. 1.5 gms of NH₂-TEG-t-butyl ester was weighed under nitrogen andtransferred to a 50 ml round bottom flask containing 25 mls of anhydrousTHF purged with N₂. After 30 mins, 1.2 moles of succinic anhydride wasadded slowly to the reaction mixture. Reaction mixture was allowed toreact overnight under the nitrogen atmosphere. The progress of thereaction was monitored by TLC (Methanol/Ethyl Acetate 1/10). After thecompletion of the reaction, the solvent was evaporated and the oilyresidue was then purified using column chromatography. Various fractionswhich seemed similar in TLC were pooled together and the productobtained was characterized by NMR. NMR indicated the presence of pureproduct (FIG. 8E).

Purification of Peptide

FIG. 7C is the synthetic scheme for peptide synthesis. Peptide wassynthesized using solid phase peptide synthesizer on a trityl chlorideresin. The purification of peptide was done by recrystallization andextraction of the peptide in methylene chloride and water. 10 mg ofpeptide was dissolved in methylene chloride was extracted with water.This extraction was repeated several times followed by the drying ofwater using methanol. Once water was dried off the solution was thenused for MALDI analysis. The MALDI analysis gave the pure product withminute amount of impurities (FIG. 8F).

Synthesis of USPIO-TEG-t-butyl Ester

30 mg of HOOC-TEG-t-butyl ester was mixed with 15.7 mg of EDC in 1 ml ofMES buffer and the reaction mixture was heated at 50° C. for about 15-20mins. This mixture was then added to the suspension of 13 mg ofUSPIO-amine in 3 mls of water and the reaction mixture was shaken on theshaker for 12 hours. After 12 hours the reaction mixture was centrifugedand washed with water and the solid was separated using magnet and keptin lypholyzer.

Synthesis of USPIO-TEG-Peptide

15 mg of USPIO-TEG t-butyl ester was first hydrolyzed with mixture ofmethylene chloride and TFA (50:50) for about 2 hours and the solvent wasevaporated. After the evaporation of the solvent the sample was kept inlypholyzer for further elemental analysis using EDS. Part of thematerial (USPIO-TEG)-15 mg was mixed with 15.7 mg of EDC in 1 ml of MESbuffer and the reaction mixture was heated at 50° C. for about 15-20mins. To this mixture 50 mgs of peptide was then added along withacetonitrile to make the peptide more soluble into the reactionsolution. The reaction mixture was shaken on the shaker for 12 hours.After 12 hours the reaction mixture was centrifuged and washed withwater and the solid was separated using magnet and kept in lypholyzer.

Synthesis of USPIO-TEG-Peptide-mPEG₅₀₀₀

25 mg of USPIO-TEG-peptide was mixed with 23.7 mg of EDC in 1 ml of MESbuffer and the reaction mixture was heated at 50° C. for about 15-20mins. To the reaction mixture 50 mg of PEG-NH₂ was then added along with1 ml of water. The reaction mixture was shaken on the shaker for 12hours. After 12 hours the reaction mixture was centrifuged and washedwith water and the solid was separated using magnet and kept inlypholyzer. Finally the USPIO-TEG-Peptide-PEG was totally deprotected bystirring the in the solution of 1:1 [TFA:CH₂Cl₂] for 4 hours. Thesolvent was evaporated and the product obtained was washed withmethylene chloride and reprecipitated using a very powerful magnet whilethe solution was decanted. This procedure was repeated 3 times so as tomake sure that all the impurities are being washed out. FIGS. 7D-7E aresynthetic schema for the synthesis of USPIO-TEG-Peptide-mPEG₅₀₀₀.

EXAMPLE 5 Dual Functioning Nanoparticles

A dual magnetic resonance imaging (MRI) contrast agent and nonviral genedelivery system comprises iron oxide gadolinium (Gd[III])-dopednanoparticles, coated with an inert layer of gold (Au) are conjugated toa novel biodegradable cationic polymer disulfide-reducible, linear,L-lysine-modified copolymers (LLC). The cationic nanoparticles aretransfect mesenchymal stem cells (MSCs) with the IL-12 gene or otheranti-tumor gene (FIG. 9).

Synthesis of Functionalized Gold-Coated Gdf[III]-Doped Iron OxideNanoparticles

Gd[III]-doped iron oxide (Fe₃O₄) nanoparticles are oxidized to Fe₂O₃ byboiling in nitric acid (HNO₃), and are then coated with gold using ahydroxylamine seedling method with chloroauric acid (HAuCl₄). Sixdifferent reaction mixtures ranging from 0 to 10% of Gd[III] ions areprepared. As can be seen from the transmission electron microscopy (TEM)image of the 4% Gd-doped nanoparticles in FIG. 10A, the synthesizedparticles are spheroidal in shape with a particle size of ˜7-10 nm. Inaddition, iron oxide nanoparticles with 0, 2, and 4% Gd[III] ions wereanalyzed with a 3.0 T Horizontal Bore General Electric Excited HDimaging system to determine if the contrast agents could be detected. Ascan be seen from FIG. 3, all of the newly synthesized nanoparticlescould be detected with MRI (copper sulfate (CuSO4) samples were used asphantom controls) (FIG. 10B). Suspended nanoparticles are reacted with100 mg of SH-PEG-NH₂ with stirring for 12 hr with methods used in ourlaboratory and purified with a magnet.

Synthesis of Au-Coated Reducible LLC Conjugated Nanoparticles withoutEthylenediamine

L-lysine HCl and CBA are added to suspended nanoparticles in (80/20 v/v)of methanol/water (MeOH/H₂O) with stirring in the dark under nitrogen.Conjugated nanoparticles are purified with a magnet. To investigate thefeasibility of the reducible LLC polymers binding DNA, linear copolymersnot conjugated to the nanoparticles were synthesized. FIG. 11 shows the¹H NMR of 25% N-boc conjugated polymer with all of the expected peaks ofthe final product.

Conjugation of N-Boc Ethylenediamine to Au-Coated Reducible LLCNanoparticles

Reducible LLC conjugated Au-coated nanoparticles suspended in water aremixed with a molar excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-boc ethylenediamine with stirringin an oil bath at 40° C. in the dark under nitrogen. Conjugatednanoparticles are purified with a magnet. The acid-liable N-boc amineprotection group present on the conjugated ethylenediamine are removedwith a (TFA)/H₂O mixture (75/25 v/v) trifluoroacetic acid.

Gel Retardation Assay

To evaluate the ability of the conjugated nanoparticles to complexplasmid DNA, a previously published gel retardation assay is performedwith a plasmid coding for the luciferase gene (pCMV-Luc). FIG. 12 showsthe synthesized LLC polymers were able to successfully condense plasmidDNA from a 25/1 N/P ratios (nitrogens of polymer/phosphates of DNA).

Reduction of Au-Coated Reducible LLC Nanoparticles

To a solution of conjugated nanoparticles, 1,4-dithio-DL-threitol (DTT)is added and the solution is incubated at room temperature. Thereduction of the disulfide bonds in the cationic polymers is monitoredwith UV spectroscopy to determine the mechanism of reaction.

Determination of T₁ and T₂ Relaxations

The longitudinal (T₁) and transverse (T₂) relaxations of the Au-coatednanoparticles are determined in agarose phantoms. Commercially purchasedFerumoxsil® (Mallinckrodt, Inc., Hazelwood, Mo.) with a crystal size of10 nm is used as a control.

Transfection Efficiency and Cytotoxicity

Cells are plated and transfected with the contrast agents, i.e.,nanoparticles and DNA with luciferase The luciferase assay is performedas previously published. Cells are plated and treated with optimizedcontrast agents and analyzed with a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayfor mitochondrial damage and a lactate dehydrogenase assay for cytosolicdamage as previously published.

MR Detection of Molecular Probes within Cells

To image MR probes within cells, MSCs treated with the optimizedcontrast agents are fixed and mixed with an agarose phantom to determinewhether or not there are differences in the T₁ and T₂ values of thenanoplexes following internalization by MSCs on the MRI scanner.

Transwell Migration Studies

The migration of transfected MSCs to plated 4T1 murine metastaticmammary breast cancer cells is studied using cells plated in a transwellcell culture dish. Cells are analyzed with microscopy.

In Vivo Mouse Model

A metastatic mouse model based on 4T1 cells injected subcutaneously inBalb/c mice is established as previously published. MSCs transfectedwith optimized contrast agents are injected intravenously via the tailvein after tumors have a volume of 75-120 mm³ and lung metastases havebeen detected. Animals are anesthetized and imaged as a function oftime. After this, animals are treated and imaged twice a week for threeweeks, blood is sampled and is analyzed for IL-12 expression with anenzyme-linked immunosorbent assay (ELISA). The animals are thensacrificed and tissue sections are obtained for analysis withhematoxylin and eosin.

The following references were cited herein:

-   1. Young Soo Kang et al. Chem. Mater, 8:2209-2211, 1996.-   2. Cozzoli et al. Chem Soc Rev, 35(11): 195-208, 2006.-   3. Pankhurst et al. J. Phys. D: Appl. Phys. 36:R167, 2003.-   4. West, J. L. and Halas, N. J. CurrOpin Biotechnol, 11(2):215-7,    2000.-   5. Yu et al. IEEE Transactions on Magnetics, 43(6):2436-2438, 2007.-   6. Yabin Sun et al., Chem. Commun., 2006:2765-2767, 2006.-   7. Dodson, J. Gene Ther, 13:283-287, 2006.-   8. Rudge et al. J. Controlled Release, 2001(74):335-340, 2001.-   9. Mah et al., Mol. Ther. 6:106, 2002.-   10. Ito et al. J. Biosci. Bioeng, 1:100, 2005.-   11. Osaka et al. Anal. Bioanal. Chem, 384:593, 2006.-   12. Dobson, J., Gene Ther 13:283, 2006.-   13. Wang Y X, H. S., Krestin G P, Eur Radiol, 11:2319-2331, 2001.-   14. Pilgrimm, H., 2003, USA, 6638494-   15. LaConte et al. Journal of Magnetic Resonance Imaging,    26:1634-1641, 2007.-   16. Drake et al. Journal of Material Chemistry, 17:4914-4918, 2007.-   17. Hong et al. Chemistry Letters, 33(11):1468-1469, 2004.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. Changes therein and other uses which are encompassedwithin the spirit of the invention as defined by the scope of the claimswill occur to those skilled in the art.

What is claimed is:
 1. A biomimetic contrast agent, comprising: anamine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core;a targeting ligand attached to the nanoparticle core via a linker; aninert outer layer of a hydrophilic polymer conjugated to the targetingligand, wherein the linker comprises a triethylene glycol polymer orpolyethylene glycol polymer.
 2. The biomimetic contrast agent of claim1, further comprising a metal doping agent in the nanoparticle core. 3.The biomimetic contrast agent of claim 2, wherein the metal doping agentis gadolinium, manganese, cobalt, or ruthenium.
 4. The biomimeticcontrast agent of claim 1, further comprising a gold coating on thenanoparticle core.
 5. The biomimetic contrast agent of claim 1, whereinthe ratio of the iron (II) to the iron(III) in the nanoparticle core isabout 1:1.
 6. The biomimetic contrast agent of claim 1, wherein thetargeting ligand comprises a contiguous peptide sequence of an enzymecleavable sequence and a targeting sequence in tandem.
 7. The biomimeticcontrast agent of claim 6, further comprising a blocking agent bound toa terminal sidechain amino acid of the peptides.
 8. The biomimeticcontrast agent of claim 6, wherein the enzyme cleavable sequence iscleavable by an enzyme associated with ovarian or breast cancer cells.9. The biomimetic contrast agent of claim 6, wherein the enzymecleavable sequence is a metalloproteinase-13 cleavable sequence.
 10. Thebiomimetic contrast agent of claim 9, wherein the MMP-13 cleavablesequence is shown in SEQ ID NO:
 1. 11. The biomimetic contrast agent ofclaim 6, wherein the targeting sequence is a endothelin-1 (ET-1)sequence, an integrin-binding sequence or a human immunodeficiency virusTat peptide sequence.
 12. The biomimetic contrast agent of claim 11,wherein the the integrin-binding sequence is shown in SEQ ID NO:
 3. 13.The biomimetic contrast agent of claim 1, wherein the inert outer layercomprises a polyethylene glycol polymer.
 14. A kit comprising thebiomimetic contrast agent of claim 1.